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CDK6 levels regulate quiescence exit in human hematopoietic stem cells.

Laurenti E, Frelin C, Xie S, Ferrari R, Dunant CF, Zandi S, Neumann A, Plumb I, Doulatov S, Chen J, April C, Fan JB, Iscove N, Dick JE - Cell Stem Cell (2015)

Bottom Line: Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation.Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function.Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system.

View Article: PubMed Central - PubMed

Affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada. Electronic address: el422@cam.ac.uk.

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LT- and ST-HSCs Are Equally Quiescent, but upon Mitogenic Stimulation They Differ in the Duration of Divisions Starting from G0 or G1(A and B) Proportion of human CB HSC and progenitor cells in each phase of the cell cycle. Parameters were assessed by flow cytometry using Ki67 and Hoechst (Ki67− 2n DNA content, G0; Ki67+ 2n DNA content, G1; Ki67+ > 2n DNA content, S-G2-M). (A) Representative flow cytometry cell cycle profiles of CB LT- and ST-HSCs and the percentage of cells in each gate. Event count: LT-HSCs (top panel), 1,320 cells; ST-HSCs (bottom panel), 1,143 cells. (B) Mean ± SEM is shown; n = 3 CB samples.(C) Cell diameter of indicated populations measured with ImageJ from microscopy pictures. n > 323 cells from four independent CB samples.(D) Mitochondrial mass as measured by flow cytometry with MitoGreen. MFI, Mean Fluorescence Intensity; mean ± S.E.M shown, n = 2 independent CB samples.(E) PhosphoS6 protein levels as measured by flow cytometry. Left panels: representative flow cytometry plots; black line, LT-HSCs; red line, ST-HSCs. Right panel: median fluorescence intensity of phosphoS6 staining. Mean ± SEM is shown. n = 2 CB samples.(F) Percentage of cells positive for phosphoRB (S807/S811) as measured by flow cytometry; mean ± S.E.M shown, n = 2 independent CB samples. GMP, granulocyte-monocyte progenitors.(G) Cumulative first division kinetics (excluding dead cells) of LT-HSCs (black) and ST-HSCs (red) from a representative CB example. Curve is least-squares sigmoid fit. R2 > 0.99. Arrowheads represent time to first division as estimated from sigmoid fit (tFirstDiv = logEC50). Time 0 is the time of exposure to mitogenic stimulus.(H) Mean time to first division (in hours).(I) Mean time of cell cycle transit (tSecondDiv = logEC50 of sigmoid fit of cumulative second division kinetics; see Figure S2E).(J) Mean time of G0 exit (in hours) (tG0exit = tFirstDiv – tSecondDiv).In (H)–(J), individual CB samples are shown; gray lines connect LT-HSC and ST-HSC parameters from the same CB. ∗∗p < 0.05, ∗∗∗p < 0.01 by paired t test. See also Figure S2.
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fig2: LT- and ST-HSCs Are Equally Quiescent, but upon Mitogenic Stimulation They Differ in the Duration of Divisions Starting from G0 or G1(A and B) Proportion of human CB HSC and progenitor cells in each phase of the cell cycle. Parameters were assessed by flow cytometry using Ki67 and Hoechst (Ki67− 2n DNA content, G0; Ki67+ 2n DNA content, G1; Ki67+ > 2n DNA content, S-G2-M). (A) Representative flow cytometry cell cycle profiles of CB LT- and ST-HSCs and the percentage of cells in each gate. Event count: LT-HSCs (top panel), 1,320 cells; ST-HSCs (bottom panel), 1,143 cells. (B) Mean ± SEM is shown; n = 3 CB samples.(C) Cell diameter of indicated populations measured with ImageJ from microscopy pictures. n > 323 cells from four independent CB samples.(D) Mitochondrial mass as measured by flow cytometry with MitoGreen. MFI, Mean Fluorescence Intensity; mean ± S.E.M shown, n = 2 independent CB samples.(E) PhosphoS6 protein levels as measured by flow cytometry. Left panels: representative flow cytometry plots; black line, LT-HSCs; red line, ST-HSCs. Right panel: median fluorescence intensity of phosphoS6 staining. Mean ± SEM is shown. n = 2 CB samples.(F) Percentage of cells positive for phosphoRB (S807/S811) as measured by flow cytometry; mean ± S.E.M shown, n = 2 independent CB samples. GMP, granulocyte-monocyte progenitors.(G) Cumulative first division kinetics (excluding dead cells) of LT-HSCs (black) and ST-HSCs (red) from a representative CB example. Curve is least-squares sigmoid fit. R2 > 0.99. Arrowheads represent time to first division as estimated from sigmoid fit (tFirstDiv = logEC50). Time 0 is the time of exposure to mitogenic stimulus.(H) Mean time to first division (in hours).(I) Mean time of cell cycle transit (tSecondDiv = logEC50 of sigmoid fit of cumulative second division kinetics; see Figure S2E).(J) Mean time of G0 exit (in hours) (tG0exit = tFirstDiv – tSecondDiv).In (H)–(J), individual CB samples are shown; gray lines connect LT-HSC and ST-HSC parameters from the same CB. ∗∗p < 0.05, ∗∗∗p < 0.01 by paired t test. See also Figure S2.

Mentions: The increased frequency of ST-HSC divisions may be due to (1) more cells actively cycling at any time, (2) increased sensitivity, or (3) faster response to mitogenic stimulation. To resolve the basis for these increased divisions, we investigated the proportions of LT- and ST-HSCs in each cell cycle phase and found them to be identical at all points during the xenotransplantation process (Figure S1B). Freshly isolated from CB, both HSC subsets had more than 90% of cells in G0 (Ki67− 2n DNA content, Figures 2A and 2B). Importantly, no cell was found in S-G2-M as determined by DNA content (Figures 2A and 2B) and by complete absence of the mitotic marker phosphoH3 (Figure S2A). Cell diameters were equally small in LT- and ST-HSCs (Figure 2C), with both lacking in cytoplasm. Metabolically, both LT- and ST-HSCs showed low mitochondrial activity (Figure 2D) and similar levels of mTOR activation (assessed by phosphoS6 staining; Figure 2E). All these parameters indicate a G0 quiescent state. To exclude a possible differential G1 arrest state for LT- and ST-HSCs, we analyzed the phosphorylation state of retinoblastoma protein (RB) at S807/S811, a marker upregulated in G0 cells before entry into G1 (Ren and Rollins, 2004). Both cell types were negative (Figures 2F and S2B). In contrast, granulocyte-monocyte progenitors (GMPs) were largely in G1, as most cells were Ki67+ with 2n DNA content (Figure 2B) and had a larger diameter (Figure 2C), visible cytoplasm, increased mitochondrial activity (Figure 2D), and RB phosphorylation on S807/S811 (Figure 2F). Collectively, these data establish that both human LT- and ST-HSCs freshly isolated from CB reside in a G0 quiescent state lacking all markers of G1.


CDK6 levels regulate quiescence exit in human hematopoietic stem cells.

Laurenti E, Frelin C, Xie S, Ferrari R, Dunant CF, Zandi S, Neumann A, Plumb I, Doulatov S, Chen J, April C, Fan JB, Iscove N, Dick JE - Cell Stem Cell (2015)

LT- and ST-HSCs Are Equally Quiescent, but upon Mitogenic Stimulation They Differ in the Duration of Divisions Starting from G0 or G1(A and B) Proportion of human CB HSC and progenitor cells in each phase of the cell cycle. Parameters were assessed by flow cytometry using Ki67 and Hoechst (Ki67− 2n DNA content, G0; Ki67+ 2n DNA content, G1; Ki67+ > 2n DNA content, S-G2-M). (A) Representative flow cytometry cell cycle profiles of CB LT- and ST-HSCs and the percentage of cells in each gate. Event count: LT-HSCs (top panel), 1,320 cells; ST-HSCs (bottom panel), 1,143 cells. (B) Mean ± SEM is shown; n = 3 CB samples.(C) Cell diameter of indicated populations measured with ImageJ from microscopy pictures. n > 323 cells from four independent CB samples.(D) Mitochondrial mass as measured by flow cytometry with MitoGreen. MFI, Mean Fluorescence Intensity; mean ± S.E.M shown, n = 2 independent CB samples.(E) PhosphoS6 protein levels as measured by flow cytometry. Left panels: representative flow cytometry plots; black line, LT-HSCs; red line, ST-HSCs. Right panel: median fluorescence intensity of phosphoS6 staining. Mean ± SEM is shown. n = 2 CB samples.(F) Percentage of cells positive for phosphoRB (S807/S811) as measured by flow cytometry; mean ± S.E.M shown, n = 2 independent CB samples. GMP, granulocyte-monocyte progenitors.(G) Cumulative first division kinetics (excluding dead cells) of LT-HSCs (black) and ST-HSCs (red) from a representative CB example. Curve is least-squares sigmoid fit. R2 > 0.99. Arrowheads represent time to first division as estimated from sigmoid fit (tFirstDiv = logEC50). Time 0 is the time of exposure to mitogenic stimulus.(H) Mean time to first division (in hours).(I) Mean time of cell cycle transit (tSecondDiv = logEC50 of sigmoid fit of cumulative second division kinetics; see Figure S2E).(J) Mean time of G0 exit (in hours) (tG0exit = tFirstDiv – tSecondDiv).In (H)–(J), individual CB samples are shown; gray lines connect LT-HSC and ST-HSC parameters from the same CB. ∗∗p < 0.05, ∗∗∗p < 0.01 by paired t test. See also Figure S2.
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fig2: LT- and ST-HSCs Are Equally Quiescent, but upon Mitogenic Stimulation They Differ in the Duration of Divisions Starting from G0 or G1(A and B) Proportion of human CB HSC and progenitor cells in each phase of the cell cycle. Parameters were assessed by flow cytometry using Ki67 and Hoechst (Ki67− 2n DNA content, G0; Ki67+ 2n DNA content, G1; Ki67+ > 2n DNA content, S-G2-M). (A) Representative flow cytometry cell cycle profiles of CB LT- and ST-HSCs and the percentage of cells in each gate. Event count: LT-HSCs (top panel), 1,320 cells; ST-HSCs (bottom panel), 1,143 cells. (B) Mean ± SEM is shown; n = 3 CB samples.(C) Cell diameter of indicated populations measured with ImageJ from microscopy pictures. n > 323 cells from four independent CB samples.(D) Mitochondrial mass as measured by flow cytometry with MitoGreen. MFI, Mean Fluorescence Intensity; mean ± S.E.M shown, n = 2 independent CB samples.(E) PhosphoS6 protein levels as measured by flow cytometry. Left panels: representative flow cytometry plots; black line, LT-HSCs; red line, ST-HSCs. Right panel: median fluorescence intensity of phosphoS6 staining. Mean ± SEM is shown. n = 2 CB samples.(F) Percentage of cells positive for phosphoRB (S807/S811) as measured by flow cytometry; mean ± S.E.M shown, n = 2 independent CB samples. GMP, granulocyte-monocyte progenitors.(G) Cumulative first division kinetics (excluding dead cells) of LT-HSCs (black) and ST-HSCs (red) from a representative CB example. Curve is least-squares sigmoid fit. R2 > 0.99. Arrowheads represent time to first division as estimated from sigmoid fit (tFirstDiv = logEC50). Time 0 is the time of exposure to mitogenic stimulus.(H) Mean time to first division (in hours).(I) Mean time of cell cycle transit (tSecondDiv = logEC50 of sigmoid fit of cumulative second division kinetics; see Figure S2E).(J) Mean time of G0 exit (in hours) (tG0exit = tFirstDiv – tSecondDiv).In (H)–(J), individual CB samples are shown; gray lines connect LT-HSC and ST-HSC parameters from the same CB. ∗∗p < 0.05, ∗∗∗p < 0.01 by paired t test. See also Figure S2.
Mentions: The increased frequency of ST-HSC divisions may be due to (1) more cells actively cycling at any time, (2) increased sensitivity, or (3) faster response to mitogenic stimulation. To resolve the basis for these increased divisions, we investigated the proportions of LT- and ST-HSCs in each cell cycle phase and found them to be identical at all points during the xenotransplantation process (Figure S1B). Freshly isolated from CB, both HSC subsets had more than 90% of cells in G0 (Ki67− 2n DNA content, Figures 2A and 2B). Importantly, no cell was found in S-G2-M as determined by DNA content (Figures 2A and 2B) and by complete absence of the mitotic marker phosphoH3 (Figure S2A). Cell diameters were equally small in LT- and ST-HSCs (Figure 2C), with both lacking in cytoplasm. Metabolically, both LT- and ST-HSCs showed low mitochondrial activity (Figure 2D) and similar levels of mTOR activation (assessed by phosphoS6 staining; Figure 2E). All these parameters indicate a G0 quiescent state. To exclude a possible differential G1 arrest state for LT- and ST-HSCs, we analyzed the phosphorylation state of retinoblastoma protein (RB) at S807/S811, a marker upregulated in G0 cells before entry into G1 (Ren and Rollins, 2004). Both cell types were negative (Figures 2F and S2B). In contrast, granulocyte-monocyte progenitors (GMPs) were largely in G1, as most cells were Ki67+ with 2n DNA content (Figure 2B) and had a larger diameter (Figure 2C), visible cytoplasm, increased mitochondrial activity (Figure 2D), and RB phosphorylation on S807/S811 (Figure 2F). Collectively, these data establish that both human LT- and ST-HSCs freshly isolated from CB reside in a G0 quiescent state lacking all markers of G1.

Bottom Line: Short-term (ST)-HSCs are also quiescent but contain high CDK6 protein levels that permit rapid cell cycle entry upon mitogenic stimulation.Enforced CDK6 expression in LT-HSCs shortens quiescence exit and confers competitive advantage without impacting function.Thus, differential expression of CDK6 underlies heterogeneity in stem cell quiescence states that functionally regulates this highly regenerative system.

View Article: PubMed Central - PubMed

Affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada. Electronic address: el422@cam.ac.uk.

Show MeSH
Related in: MedlinePlus